Photoresponse drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a photoresponse drug delivery system and a preparation method and application thereof. The photoresponse drug delivery carrier comprises a photosensitive liposome and a metal polyphenol coordination polymer micelle, the metal polyphenol coordination polymer micelle comprises a metal polyphenol network coating and a polymer micelle, the surface of the polymer micelle is coated with the metal polyphenol network coating, and the metal polyphenol network coating is coated with the polymer micelle. The metal polyphenol coordination polymer micelle is loaded on the surface of the photosensitive liposome; the photosensitive lipidosome comprises a lipidosome membrane and an inner water phase located in the lipidosome membrane, the inner water phase comprises EDTA and ammonium salt, and the lipidosome membrane comprises a lipid material and a photosensitizer. The photoresponse drug delivery system has an efficient photoresponse drug release rate, sequential release of drugs can be achieved, and multi-mode combined anti-tumor is achieved through combination of photodynamic and chemotherapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a photoresponsive drug delivery system, its preparation method, and its uses. Background Technology

[0002] Currently, combination therapy with two or more drugs has become a routine approach to improving clinical outcomes. Countries around the world actively support the development of fixed-dose combination drugs, which combine two or more individually active drug components into a single-dose formulation. This leverages the different mechanisms of action of the drugs to produce synergistic therapeutic effects. Related industry development guidelines have been published to better regulate and guide research on the combined use of multiple drugs.

[0003] With the increasing penetration of nanotechnology into the biomedical sciences, nano-form drug delivery has emerged, especially for tumor-specific drug delivery. Nanocarrier-based drug delivery systems can enhance drug accumulation within tumors by targeting specific tumor sites, while reducing drug distribution in normal tissues and cells, thus lowering drug toxicity and side effects. This offers potential advantages in combination anti-tumor therapy. Vyxeos, approved by the FDA in 2017 for the treatment of acute myeloid leukemia, is a combination formulation encapsulating daunorubicin and cytarabine in a 1:5 molar ratio within liposomes. Compared to combination therapy with free drugs (patient survival 5.9 months), this dual-drug co-delivery liposomal formulation extends overall survival to 9.6 months.

[0004] Various drug delivery systems already exist on the market that deliver two or more drug molecules with synergistic pharmacological activities to tumor cells. However, tumor development and progression are complex and heterogeneous. The tumor microenvironment is composed not only of tumor cells but also includes the tumor extracellular matrix, connective cells, tissues, and various immune cells. Therefore, in the field of drug delivery, in addition to targeted delivery to tumor cells, it is also necessary to consider delivering drugs to other cells in the tumor microenvironment that play an important role in tumor development, and to combine them with novel drug targets for tumor therapy. Thus, when the drug targets for co-administration are located both inside tumor cells (e.g., intracellular microtubules and the nucleus) and outside tumor cells (e.g., extracellular domains of tumor cell membrane proteins, tumor stromal cells, extracellular matrix, etc.), developing a sequential delivery strategy for active drugs would be an optimal choice for co-drug delivery.

[0005] A major challenge in sequential drug delivery system design is that drugs targeting extracellular tumor cells must be completely unloaded before the nanocarrier carrying intracellular drugs is taken up into the tumor cells. Current nanodelivery strategies largely rely on enzymes highly expressed in tumors or the acidic tumor microenvironment to develop corresponding drug release mechanisms. Generally, nanomedicines exhibit good stability in the bloodstream. When they reach tumor tissue or the vicinity of cells, the nanocarrier responds to highly expressed proteases (such as hyaluronidase, matrix metalloproteinases, and cathepsins) or the acidic environment of the tumor microenvironment, resulting in changes in the physicochemical properties of the nanocarrier, such as surface charge reversal, carrier particle size changes, and surface ligand exposure, thereby triggering the release of the active drug targeting extracellular tumor cells. In a previous report, doxorubicin (DOX), an intracellular drug, was encapsulated in a nanocarrier modified with a cationic transmembrane peptide (R8H3). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an extracellular drug, was electrostatically adsorbed onto the surface of the nanocarrier. The nanocarrier was then encapsulated with hyaluronic acid, resulting in the nanodrug (TRAIL / Dox-Gelipo). After the nanodrug is targeted to the tumor site via enhanced permeation retention (EPR) effects, hyaluronidase, highly expressed in the tumor microenvironment, hydrolyzes hyaluronic acid, releasing TRAIL. TRAIL then acts on death receptors on the tumor cell membrane, inducing tumor cell death and achieving extracellular delivery of the tumor. Following hyaluronic acid hydrolysis, the cationic transmembrane peptide is exposed, and the surface charge of the nanocarrier becomes positive, adsorbing onto the negatively charged tumor cell membrane. This mediates the entry of the DOX-carrying nanocarrier into the tumor cell, achieving intracellular delivery of the tumor.

[0006] However, the enzyme- or acid-dependent disintegration of nanocarriers is a time-consuming process, and this endogenous stimulation is susceptible to tumor heterogeneity, resulting in suboptimal drug release efficiency. Typically, it takes 1.5–8 hours to release 50% of the drug targeting the extracellular tumor cells, while complete drug release usually requires 24–48 hours. In this situation, a significant proportion of the drug targeting the extracellular tumor cells is inadvertently delivered into the tumor cells along with the nanocarrier. Therefore, the potential of combination therapy may not be fully realized.

[0007] Therefore, there is an urgent need for a drug delivery system that is independent of tumor heterogeneity and can rapidly disintegrate and release drugs. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a photoresponsive drug delivery system, its preparation method, and its uses.

[0009] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0010] A first aspect of the present invention protects a photoresponsive drug delivery carrier comprising photosensitive liposomes and metal polyphenol coordination polymer micelles, wherein the metal polyphenol coordination polymer micelles comprise a metal polyphenol network coating and polymer micelles, and the metal polyphenol network coating is coated on the surface of the polymer micelles.

[0011] The metal polyphenol coordination polymer micelles are loaded on the surface of the photosensitive liposomes;

[0012] The photosensitive liposome comprises a liposome membrane and an internal aqueous phase located within the liposome membrane, the internal aqueous phase comprising ethylenediaminetetraacetic acid and an ammonium salt, and the liposome membrane comprising a lipid material and a photosensitizer.

[0013] A second aspect of this invention protects a method for preparing a photoresponsive drug delivery carrier as described above, comprising the following steps:

[0014] The photosensitive liposomes and metal polyphenol coordination polymer micelles self-assemble to form the photoresponsive drug delivery carrier.

[0015] A third aspect of the present invention protects a photoresponsive drug delivery system comprising a photoresponsive drug delivery carrier and an antitumor drug as described above, wherein the antitumor drug comprises a first drug or a second drug, and the photosensitive liposome encapsulates the first drug or the polymer micelles load the second drug.

[0016] The fourth aspect of this invention protects the use of the photoresponsive drug delivery carrier or the photoresponsive drug delivery system described above in the preparation of antitumor drugs.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1) In the photoresponsive drug delivery carrier of the present invention, both photosensitive liposomes and polymeric micelles can be loaded with drugs individually or simultaneously, and can simultaneously load two drugs with different activities, such as encapsulating the first drug in the photosensitive liposomes and loading the second drug in the polymeric micelles; it has photoresponsive characteristics and can overcome the limitations of relying on highly expressed enzymes or acidic microenvironments in tumor tissue to achieve drug release from nanocarriers, and is no longer affected by the heterogeneous tumor microenvironment.

[0019] 2) The photoresponsive drug delivery carrier of this application has a high drug loading capacity and encapsulation efficiency. The encapsulation efficiency of the drug by the photosensitive liposome is 80-95%, and the drug loading capacity of the polymer micelle is 1-3%.

[0020] 3) The photoresponsive drug delivery carrier of this application has high photoresponsive drug release efficiency and sequential drug release capability. After near-infrared light irradiation, firstly, under the induction of photosensitizer, the permeability of the photosensitive liposome membrane increases, triggering the rapid and efficient release of the first drug acting on the extracellular target of tumor cells. The release rate of the first drug is approximately 100% within 5 minutes. Then, small-diameter polymer micelles loaded with the second drug detach from the surface of the photosensitive liposome. Due to the small particle size of the polymer micelles, they can be taken into the tumor cells through tumor cell-mediated endocytosis, realizing the release of the second drug acting on the intracellular target of tumor cells. Its release rate within 12 hours is approximately 40%.

[0021] 4) The photoresponsive drug delivery carrier of this application exhibits good serum stability and storage stability. The photoresponsive drug delivery system of this application did not aggregate or disintegrate after being stored in PBS containing 10% FBS for 24 hours, demonstrating good serum stability. Furthermore, the photoresponsive drug delivery system of this application did not aggregate or disintegrate after being stored in PBS for 7 days, demonstrating good storage stability.

[0022] 5) The photoresponsive drug delivery system of this application, loaded with the first drug and the second drug, showed in HUVEC tubule formation and migration experiments after near-infrared light irradiation that the first drug directly acts on the extracellular target (vascular endothelial cells) of tumor cells to achieve enhanced anti-tumor angiogenesis ability; the tubule formation and migration experiments of A2058 tumor cells proved that the polymer micelles loaded with the second drug released in a photoresponsive manner can produce enhanced anti-angiogenic mimicry effect.

[0023] 6) After administration of the photoresponsive drug delivery system of this application into the body, it can effectively inhibit tumor growth and prolong survival; near-infrared light can significantly inhibit tumor growth with an inhibition rate of over 80%, and can also significantly prolong the survival time of mice to over 63.1%.

[0024] 7) The photoresponsive drug delivery system of this application, through the organic combination of anti-angiogenesis and anti-VM, relieves the inhibition of VM under the effect of anti-tumor angiogenesis, thereby improving the anti-tumor effect. Attached Figure Description

[0025] Figure 1 The diagram shown is a schematic diagram of the structure of the photoresponsive drug delivery carrier in Embodiment 1 of this application.

[0026] Figure 2 The image shows the fluorescence signals of DAS-NP (DiO) micelles labeled with metal polyphenol coordination polymers (DiO), Lipo (DiI) labeled with photosensitive liposome bilayers, and a physical mixture of the two ((DiO) + (DiI)) in Example 1 of this application.

[0027] Figure 3The image shown represents the deep penetration results of the photoresponsive drug delivery carrier into the tumor sphere in Example 1 of this application. In this image, A represents the results of examining coumarin 6@Lipo (660nm, 300mW / cm²) with or without light pretreatment. 2 The penetration ability of A2058 tumor spheres (5 min); B is the quantitative fluorescence intensity at the dashed line in Figure A.

[0028] Figure 4 The diagram shown is a schematic diagram of the preparation method of the photoresponsive drug delivery system (MS@Lipo) in Embodiment 2 of this application.

[0029] Figure 5 The image shown is a photoresponse result diagram of the photoresponsive drug delivery system (MS@Lipo) in Embodiment 3 of this application. Wherein, A represents different concentrations of EDTA and Fe... 3+ The UV-Vis absorption spectrum of the reaction shows a characteristic absorption peak at 258 nm; B represents the release of EDTA from MS@Lipo after irradiation; C represents the particle size change of MS@Lipo before and after irradiation as detected by Nanosight; D represents the particle size change of MS@Lipo before and after irradiation as detected by DLS.

[0030] Figure 6 The image shown is a photoresponse result diagram of the photoresponsive drug delivery system (MS@Lipo) formed by photosensitive liposomes without EDTA in the inner water cavity in Example 3 of this application.

[0031] Figure 7 The image shows the photoresponsive drug release from the photoresponsive drug delivery system (MS@Lipo) in Embodiment 3 of this application. In this image, A represents the release of dasatinib from MS@Lipo; and B represents the release of sunitinib from MS@Lipo.

[0032] Figure 8 The images shown are representative photographs of crystal violet staining of migrating HUVECs after co-incubation of the S@Lipo photoresponsive drug delivery system (with or without light pretreatment) with HUVECs in Example 4 of this application; and graphs showing the migration rates of HUVECs at different sunitinib concentrations in S@Lipo (with or without light pretreatment).

[0033] Figure 9The image shows the effect of the photoresponsive drug delivery system S@Lipo in Example 5 of this application on HUVEC tubule formation. A represents a representative photograph of HUVEC tubule formation after incubation with S@Lipo (5, 10, 20 μM sunitinib) without photopretreatment; B represents a representative photograph of HUVEC tubule formation after incubation with S@Lipo (5, 10, 20 μM sunitinib) under photopretreatment; C represents a representative photograph of HUVEC tubule formation in the control group; and D quantifies the tubule formation rate under different sunitinib concentrations (5, 10, 20 μM) in S@Lipo with or without photopretreatment.

[0034] Figure 10 The figure shown is a graph illustrating the effect of the photoresponsive drug delivery system M@Lipo on the migration ability of A2058 cells in Example 5 of this application. Figure A shows the effect of M@Lipo with or without light pretreatment on the migration ability of A2058 cells observed using IncuCyte; Figure B shows the quantitative analysis of cell healing ability in Figure A.

[0035] Figure 11 The image shown is a representative photograph and a quantitative result of the tube formation rate of A2058 after the photoresponsive drug delivery system M@Lipo and A2058 were co-incubated in Example 5 of this application.

[0036] Figure 12 The figure shown is a graph illustrating the antitumor effect of the photoresponsive drug delivery system (MS@Lipo) in a melanoma model mouse as described in Example 6 of this application. In the graph, A is a schematic diagram of the treatment regimen; B is the tumor growth curve; C is the mouse survival curve; and D is the mouse body weight. Detailed Implementation

[0037] A first aspect of the present invention protects a photoresponsive drug delivery carrier comprising photosensitive liposomes and metal polyphenol coordination polymer micelles, wherein the metal polyphenol coordination polymer micelles comprise a metal polyphenol network coating and polymer micelles, and the metal polyphenol network coating is coated on the surface of the polymer micelles.

[0038] The metal polyphenol coordination polymer micelles are loaded on the surface of the photosensitive liposomes;

[0039] The photosensitive liposome comprises a liposome membrane and an internal aqueous phase located within the liposome membrane, the internal aqueous phase comprising ethylenediaminetetraacetic acid and an ammonium salt, and the liposome membrane comprising a lipid material and a photosensitizer.

[0040] In some embodiments, the particle size of the photoresponsive drug delivery carrier is 130-200 nm, or it can be 130-160 nm, 150-190 nm, 185-200 nm, or 175 nm, 180 nm, or 185 nm.

[0041] In some embodiments, the mass ratio of the photosensitive liposomes, the metal polyphenol network coating, and the polymer micelles is (0.1–0.8):(0.05–0.4):1, or it can be (0.1–0.8):(0.05–0.18):1, (0.1–0.8):(0.15–0.26):1, (0.1–0.8):(0.21–0.4):1, or 0.5:0.2:1.

[0042] The photoresponsive drug delivery carrier of this application uses a photosensitive liposome as its core, with polymer micelles adhering to the outer surface of the core through a metal polyphenol network coating to form a satellite structure. It encapsulates ethylenediaminetetraacetic acid (EDTA) and a photosensitizer within the water lumen of the photosensitive liposome. Upon light irradiation, the photosensitizer induces an increase in the permeability of the liposome membrane, causing the EDTA encapsulated in the water lumen to be released. The EDTA then competitively chelates with metal ions in the metal polyphenol network coating, thereby disrupting the coating and causing the polymer micelles to detach from the surface of the photosensitive liposome. The detached polymer micelles are then taken up into the cell via cell-mediated endocytosis and penetrate deep into the tumor. The photoresponsive drug delivery carrier of this application possesses photoresponsive properties, overcoming the limitations of relying on highly expressed enzymes or acidic microenvironments in tumor tissue to achieve drug release via nanocarriers, and is no longer affected by the heterogeneous tumor microenvironment. The photoresponsive drug delivery carrier of this invention can simultaneously load drugs in photosensitive liposomes and polymer micelles, achieving combined therapy and avoiding the limitation of drug efficacy in vivo due to off-target distribution at the pharmacokinetic level and off-target effects at the pharmacodynamic level. When the photoresponsive drug delivery carrier of this invention is irradiated with light, the liposome membrane ruptures, releasing the drug loaded in the photosensitive liposome. Simultaneously, the metal binding in the EDTA and metal polyphenol network coating further releases the polymer micelles, and the released polymer micelles are more likely to penetrate deeper into the tumor, thereby releasing the drug loaded in the polymer micelles, enabling sequential drug delivery.

[0043] Using A2058 tumor spheres as an example, the coumarin 6-labeled photoresponsive drug delivery carrier not only exhibited clear red fluorescence signals on the periphery of the tumor spheres but also inside them. Furthermore, after light treatment, significant red fluorescence signals were observed within a depth of 100-300 μm in the tumor spheres. The inventors also developed a photoresponsive drug delivery carrier for photosensitive liposomes without EDTA in the water cavity. After light irradiation, the particle size of the carrier did not change significantly, indicating that even after light irradiation, it could not achieve the disintegration of the photosensitive liposomes and the detachment of polymer micelles.

[0044] In this application, the photoresponsive drug delivery carrier can carry drugs individually or simultaneously using photosensitive liposomes and polymeric micelles. It can also simultaneously load two drugs with different activities, which is beneficial for achieving combined treatment of multiple drugs and can provide an effective carrier for improving clinical treatment effects.

[0045] In addition, the photoresponsive drug delivery carrier of this application has a high efficiency of photoresponsive drug release, and the drug loaded in the photosensitive liposome can be 100% released within 5 minutes. It also has sequential drug release performance, that is, the drug loaded in the photosensitive liposome is released first, and the drug loaded in the polymer micelle is released later.

[0046] This application's photoresponsive drug delivery carrier utilizes the phenolic hydroxyl groups of polyphenols to chelate with metal ions. When multiple phenolic hydroxyl groups are present, they can simultaneously chelate with metal ions, forming a three-dimensional network. This property allows metal polyphenols to be coated onto the surface of polymer micelles. Simultaneously, polyphenols exhibit interfacial adhesion effects, enabling them to adsorb onto the surface of photosensitive liposomes. Therefore, the polymer micelles can be tightly connected to the photosensitive liposomes through the metal polyphenol network coating.

[0047] In some embodiments, the particle size of the photosensitive liposomes is 100-150 nm, or it can be 100-125 nm, or it can be 118-136 nm, or it can be 128-150 nm, or it can be 130 nm, 110 nm, or 120 nm.

[0048] In some embodiments, the ammonium salt is selected from one or more of ammonium sulfate, ammonium phosphate, ammonium chloride, and ammonium acetate. Preferably, it is ammonium sulfate.

[0049] In some embodiments, the photosensitizer is selected from one or more of porphyrin-phospholipids, verteporfin, hematoporphyrin derivatives, and BODIPY derivatives. In this application, the photosensitizer porphyrin-phospholipids increases the permeability of the photosensitive liposome membrane by inducing the generation of reactive oxygen species (ROS).

[0050] In some embodiments, the lipid material is selected from one or both of phospholipids and cholesterol.

[0051] In some specific embodiments, the lipid material is a mixture of phospholipids and cholesterol, and the molar ratio of phospholipids to cholesterol is (30-80):(0.1-60), or it can be (30-80):(0.1-27), or it can be (30-80):(22-45), or it can be (30-80):(38-60), or it can be, for example, 1:1.

[0052] In some specific embodiments, the phospholipid is selected from one or two of dioleoylphosphatidylcholine (DOPC), phosphocholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), and dipalmitoylphosphatidylcholine (DPPC). In some examples, it is dioleoylphosphatidylcholine (DOPC) and phosphocholine (DSPC). Preferably, the molar ratio of dioleoylphosphatidylcholine (DOPC) to phosphocholine (DSPC) is 10.75:37.25. Cholesterol is a hydrophobic molecule that can bind to phospholipids, embedding itself in the membrane, preventing phospholipids from condensing into crystalline structures, and regulating membrane fluidity. As a component of liposome membranes, cholesterol can stabilize liposomes.

[0053] In some specific embodiments, the lipid material is selected from phospholipids and cholesterol, wherein the phospholipids comprise dioleoylphosphatidylcholine (DOPC) and phosphocholine (DSPC), and the molar ratio of DOPC, DSPC, and cholesterol is 10.75:37.25:50. In some specific embodiments, DOPC, DSPC, and cholesterol are used as the liposome membrane, and the drug is loaded via EDTA using an ammonium sulfate gradient method.

[0054] In some embodiments, the molar ratio of the lipid material to the photosensitizer is (60–98):2, or it can be (60–72):2, (68–85):2, or (79–98):2. In some examples, it is 98:2.

[0055] In some embodiments, the mass ratio of the lipid material to EDTA is 1:(2-6), or 1:(2-4.2), or 1:(3-5.5), or 1:(4.2-6). In some examples, it is 1:4.

[0056] In some embodiments, the mass ratio of the lipid material to the ammonium salt is 1:(2-6), or it can be 1:(2-4.2), 1:(3-5.5), or 1:(4.2-6). In some examples, it is 1:3.3.

[0057] In some embodiments, the photosensitive liposomes are prepared using conventional photosensitive liposome preparation processes. In some instances, the photosensitive liposomes are prepared using a thin-film hydration method. Preferably, the lipid material and photosensitizer are dissolved in a first organic solvent to form a thin film, and then ammonium salt and EDTA are added for hydration to obtain the photosensitive liposomes.

[0058] More preferably, the first organic solvent is selected from one or both of methanol and chloroform, specifically a mixture of methanol and chloroform; in the mixture, the volume ratio of methanol to chloroform is 1:(1-6), or it can be 1:(1-2.8), or it can be 1:(2-5.5), or it can be 1:(4.2-6). In some examples, it is 1:3.

[0059] More preferably, the thin film is obtained by rotary evaporation at a temperature of 55–60°C, or 55–58°C, or 56–60°C. In some examples, the temperature is 60°C.

[0060] In some embodiments, the particle size of the polymer micelles is 30-50 nm, or 30-41 nm, or 38-45 nm, or 41-50 nm, or 30 nm, 35 nm, 40 nm, or 45 nm.

[0061] In some embodiments, the polymer micelles are made from polymeric materials. Polymer micelles are supramolecular structures formed by the self-assembly of amphiphilic block copolymers in aqueous solution. By attaching specific targeting ligands to the hydrophilic surface of the micelles, active targeted drug delivery is achieved using amphiphilic micelles.

[0062] In some specific embodiments, the polymer material is selected from any one or more block structure polymers formed from polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-glycolic acid), polycaprolactone (PCL), polytrimethylene carbonate, polydioxanone, polyacrylic acid, polyacrylate, and polypeptides.

[0063] Preferably, the polymer material is a block structure polymer material formed by polyethylene glycol and polycaprolactone, namely methoxy poly(ethylene glycol)-poly(ε-caprolactone). The weight-average molecular weight of the polycaprolactone is 5000, and the weight-average molecular weight of the polyethylene glycol is 5000. The applicant has also attempted to prepare polymer micelles using a weight-average molecular weight of 1000 for polycaprolactone and a weight-average molecular weight of 3000 for polyethylene glycol. However, the resulting polymer micelles were larger than 20-30 nm, which is not conducive to deep penetration into tumors and would also result in an excessively large final particle size of the drug delivery system, making it impossible to achieve tumor targeting through the EPR effect.

[0064] In some embodiments, the polymer micelles are prepared using conventional polymer micelle preparation processes. In some examples, the polymer micelles are prepared using a nanoprecipitation method. Alternatively, the polymer micelles can be prepared using thin-film dispersion and solvent evaporation methods. Preferably, the polymer material is dissolved in a second organic solvent to form an organic phase, which is then added dropwise to an aqueous phase to obtain the polymer micelles.

[0065] More preferably, the second organic solvent is selected from one or more of N,N-dimethylformamide, acetonitrile, and DMSO. In some examples, it is N,N-dimethylformamide. Those skilled in the art can select a suitable organic solvent based on the solubility of the drug and the polymeric material to obtain an organic phase.

[0066] More preferably, the aqueous phase is selected from phosphate buffer. The pH of the phosphate buffer can be 7 to 8.5. In some examples, it is 7.4. The phosphate buffer is formed from sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0067] More preferably, after the addition is complete, the process further includes dialysis or concentration. The dialysis is performed on a dialysis membrane to remove the organic solvent. The dialysis membrane has a molecular weight cutoff of 35,000 Da. The concentration includes ultrafiltration or centrifugation. The ultrafiltration tube has a molecular weight cutoff of 10,000 Da; the centrifugation speed is 2500 g.

[0068] In some embodiments, the metal polyphenol network is a three-dimensional network formed by polyvalent metal ions and polyphenolic compounds.

[0069] In some specific embodiments, the polyphenol is selected from one or more of tannic acid (TA), ellagic acid (EA), epigallocatechin gallate (EGCG), chlorogenic acid (ChA), gallic acid (GA), caffeic acid (CA), quercetin (QR), myricetin (My), luteolin (Fi), luteolin (Lu), resveratrol (Re), gossypol (Go), and proanthocyanidins (OPC). In some examples, it is tannic acid (TA).

[0070] In some specific embodiments, the multivalent metal ion is selected from one or more of Fe(III), Mn(II), Co(II), Zn(II), Ni(II), Cu(II), and Ti(IV). In some examples, it is Mn(II). The metal in the multi-metal network coating can be one, two, or multiple multivalent metal ions. The multivalent metal ions are derived from multivalent metal salts, such as manganese chloride, ferric chloride, cobalt chloride, copper chloride, and zinc chloride. In some examples, it is manganese chloride.

[0071] A second aspect of this invention protects a method for preparing a photoresponsive drug delivery carrier as described above, comprising the following steps:

[0072] The photosensitive liposomes and metal polyphenol coordination polymer micelles self-assemble to form the photoresponsive drug delivery carrier.

[0073] In some implementations, the steps of the method are as follows:

[0074] 1) Obtain polymer micelles; obtain photosensitive liposomes;

[0075] 2) The polymer micelles, multivalent metal ions, and polyphenols undergo a first reaction to obtain an intermediate product;

[0076] 3) The intermediate product, the photosensitive liposome, and the multivalent metal ions undergo a second reaction to obtain the photoresponsive drug delivery carrier.

[0077] In some embodiments, the first or second reaction further includes the addition of a promoter. The promoter is selected from 3-(N-morpholino)propanesulfonic acid (MOPS) or sodium hydroxide, specifically 3-(N-morpholino)propanesulfonic acid (MOPS). The pH value is less than 10.0 during the first or second reaction, specifically 8. This application has found that the addition of 3-(N-morpholino)propanesulfonic acid or sodium hydroxide can promote the encapsulation of the metal polyphenol network coating on the surface of the polymer micelles. The addition of MOPS in both the first and second reactions improves the encapsulation performance of the polymer micelles and the adhesion performance between the polymer micelles and the photosensitive liposomes, while reducing damage to the photosensitive liposomes or polymer micelles.

[0078] In some embodiments, the first or second reaction is carried out at room temperature and pressure.

[0079] In some embodiments, the multivalent metal ion is selected from one or more of Fe(III), Mn(II), Co(II), Zn(II), Ni(II), Cu(II), and Ti(IV); the multivalent metal ion originates from one or more of manganese salts, iron salts, cobalt salts, copper salts, and zinc salts. Examples include manganese chloride, ferric chloride, cobalt chloride, copper chloride, and zinc chloride. In some instances, manganese chloride is used.

[0080] A third aspect of the present invention protects a photoresponsive drug delivery system comprising a photoresponsive drug delivery carrier as described above and an antitumor drug, wherein the antitumor drug comprises a first drug or a second drug, and the photosensitive liposomes are encapsulated with the first drug or the polymer micelles are loaded with the second drug.

[0081] In some embodiments, the first drug is selected from drugs targeting extracellular tumor cells. The first drug is selected from anti-angiogenic drugs, wherein the encapsulation rate of the anti-angiogenic drug is 80-95%. The first drug is selected from one or more of sunitinib, sorafenib, axitinib, and lenvatinib.

[0082] In some embodiments, the second drug is selected from drugs that act on intracellular targets of tumor cells. The second drug is selected from drugs that inhibit vasculogenic mimicry (VM), i.e., anti-VM drugs, wherein the drug loading of the anti-VM drug is 1-3%. The second drug is selected from one or more of dasatinib, CVM-1118, and fasudil.

[0083] In some specific embodiments, the first drug is selected from anti-angiogenic drugs and the second drug is selected from anti-VM drugs. Highly aggressive tumor cells can meet their energy needs by deforming and remodeling the extracellular matrix to form vascular-like channels, a phenomenon known as vasculogenic mimicry (VM). VM is an endothelial-independent tumor microcirculation pattern that promotes tumor cell secretion of proteolytic enzymes by providing blood perfusion, thereby degrading the basement membrane and extracellular matrix and promoting tumor growth and metastasis. Existing anti-angiogenic drugs not only fail to counteract VM but also induce tumors to form more VM more rapidly. VM is insensitive to traditional radiotherapy and chemotherapy, thus leading to the problem of resistance to anti-angiogenic drugs caused by VM. The photoresponsive drug delivery system of this application simultaneously loads anti-tumor angiogenesis drugs and anti-VM drugs. After near-infrared light irradiation (660nm), the photosensitizer induces an increase in the permeability of the photosensitive liposome membrane, promoting the efficient release of anti-angiogenesis drugs from the photosensitive liposomes for anti-angiogenic therapy. Meanwhile, the anti-VM drugs that disintegrate from the nucleus-satellite structure can continue to penetrate deeper into the tumor to improve tumor vascular mimicry, thereby effectively overcoming the treatment resistance caused by tumor vascular mimicry in anti-tumor angiogenesis. Therefore, the photoresponsive drug delivery system of this invention has important scientific significance and research value.

[0084] In some embodiments, the photosensitive liposomes encapsulate a first drug or the polymer micelles load a second drug.

[0085] In some specific embodiments, the first drug is sunitinib. In this invention, a photoresponsive drug delivery system S@Lipo, formed by loading only sunitinib onto photosensitive liposomes and leaving polymer micelles unloaded with the second drug, was used to study HUVEC migration and tubule formation in vitro. The results showed that even without light treatment, the S@Lipo system could inhibit HUVEC migration and tubule formation. However, the inhibition rate was even better after pretreatment with light before adding HUVECs. Furthermore, the S@Lipo system containing 20 μM sunitinib, after pretreatment with light, almost completely inhibited HUVEC migration, with a migration rate of less than 5%; the S@Lipo system containing 5 μM sunitinib, after pretreatment with light, completely inhibited tubule formation. This indicates that sunitinib released from the photoresponsive drug delivery system can directly act on extracellular targets (vascular endothelial cells) of tumor cells, thereby enhancing anti-angiogenic capabilities.

[0086] In some specific embodiments, the second drug is dasatinib. In this invention, a photoresponsive drug delivery system M@Lipo, formed by loading dasatinib onto polymer micelles without loading the first drug, was used to study A2058 migration and tubule formation in vitro. The results showed that even without light treatment, the M@Lipo system could inhibit A2058 migration and tubule formation. However, the inhibition rate was even better after light pretreatment followed by the addition of A2058. Furthermore, the S@Lipo system containing 20 μM dasatinib, after light pretreatment, almost completely inhibited HUVEC migration, with a migration rate of less than 5%; the S@Lipo system containing 5 μM dasatinib, after light pretreatment, completely inhibited tubule formation. This indicates that the polymer micelles loaded with dasatinib in the photoresponsive drug delivery system can directly act on intracellular targets (tumor cells), thereby enhancing the anti-angiogenic mimicry effect.

[0087] In some specific embodiments, the first drug is sunitinib, and the second drug is dasatinib. The photoresponsive drug delivery system MS@Lipo, formed by loading sunitinib as the first drug onto photosensitive liposomes and dasatinib as the second drug onto polymer micelles, was administered to a melanoma mouse model. The results showed that both light-treated and untreated systems significantly inhibited tumor growth, but light-treated systems exhibited the strongest inhibitory effect, with a tumor inhibition rate reaching 80%. Furthermore, compared to the control group, the survival time was significantly prolonged by 63.1%. The photoresponsive drug delivery system of this invention encapsulates sunitinib, which targets extracellular tumor cells, within photosensitive liposomes and loads dasatinib, which targets intracellular tumor cells, into polymer micelles. Light irradiation induces the initial release of sunitinib from the water cavities within the photosensitive liposomes, allowing it to directly act on vascular endothelial cells. The dasatinib-loaded polymer micelles detach from the photosensitive liposomes, penetrate deeper into the tumor, and are more readily taken up by tumor cells, followed by dasatinib release. This sequential drug release—that is, sunitinib is released first, followed by dasatinib—achieves sequential drug delivery. Furthermore, this photoresponsive drug delivery system enables the combined use of drugs targeting extracellular and intracellular tumor cells, as well as photodynamic therapy, allowing for multimodal combined anti-tumor therapy.

[0088] The photoresponsive drug delivery system of this invention can improve drug release rates after light irradiation. Without light treatment, the release rates of dasatinib and sunitinib are less than 10% within 24 hours; however, after light irradiation, the release rate of sunitinib encapsulated in photosensitive liposomes is about 100% within 5 minutes, and the release rate of dasatinib loaded with polymer micelles reaches 40% within 12 hours.

[0089] In some embodiments, the photoresponsive drug delivery system delivers the drug via injection, oral administration, or topical administration.

[0090] In some embodiments, the photoresponsive drug delivery system is a combination of photodynamic and chemotherapeutic drugs.

[0091] In some implementations, pharmaceutically acceptable excipients are also included.

[0092] In some specific embodiments, the pharmaceutically acceptable excipient should be compatible with the composite material, i.e., able to be blended with it without significantly reducing the effectiveness of the photoresponsive drug delivery system under normal circumstances. In the antitumor drug described in this application, the pharmaceutically acceptable excipient is selected from one or more of carriers, diluents, binders, lubricants, and wetting agents. Specific examples of substances that can serve as pharmaceutically acceptable carriers, diluents, binders, lubricants, and wetting agents include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to aid in the stability of the formulation or to contribute to its activity or bioavailability.

[0093] This invention also protects a method for preparing the photoresponsive drug delivery system as described above, comprising the following steps: encapsulating a first drug in photosensitive liposomes, loading a second drug onto metal polyphenol coordination polymer micelles; and self-assembling the photosensitive liposomes encapsulated with the first drug and the metal polyphenol coordination polymer micelles loaded with the second drug to form the photoresponsive drug delivery system.

[0094] In some embodiments, the method includes the following steps:

[0095] 1) The polymer micelles are loaded with a second drug, or the photosensitive liposomes are loaded with a first drug;

[0096] 2) The polymer micelles loaded with the second drug, polyvalent metal ions, and polyphenols are subjected to a first reaction to obtain an intermediate product;

[0097] 3) The intermediate product, the photosensitive liposome loaded with the first drug, and the polyvalent metal ion salt undergo a second reaction to obtain the photoresponsive drug delivery system.

[0098] Preferably, the method for encapsulating the first drug with the photosensitive liposomes is as follows: mixing and incubating the photosensitive liposomes and the first drug. The mass ratio (i.e., drug-liposome ratio) of the first drug to the photosensitive liposomes is 1:(1-16). In some examples, it is 1:8.

[0099] More preferably, the incubation temperature is 55–65°C. In some examples, it is 60°C. The incubation time is 0.5–5 hours. In some examples, it is 1 hour.

[0100] More preferably, the incubation process further includes ultrafiltration to remove free first drug not loaded into the photosensitive liposomes. The ultrafiltration has a molecular weight cutoff of 10,000 Da.

[0101] Preferably, the method for loading the second drug onto the polymer micelles is as follows: dissolving the second drug and the polymer material in a second solvent to form an organic phase, and then adding it dropwise into an aqueous phase. The mass ratio of the second drug to the polymer material is 1:(10-60), specifically 1:33. The second solvent is selected from N,N-dimethylformamide, and the aqueous phase is a phosphate buffer solution with a concentration of 0.5M and a pH of 7.4.

[0102] More preferably, the ratio of the organic phase to the aqueous phase is 1:(1-10), specifically 1:4.

[0103] More preferably, the addition further includes purification to remove free second drug and second solvent (such as N,N-dimethylformamide) not loaded into the polymer micelles. The purification is performed using a dialysis membrane with an ultrafiltration molecular weight cutoff of 35,000 Da. More preferably, the purification further includes concentration. The concentration is performed using an ultrafiltration tube with an ultrafiltration molecular weight cutoff of 10,000 Da.

[0104] Further preferably, polymer micelles loaded with the second drug, polyphenols, multivalent metal ions, and a promoter are mixed in water to encapsulate a metal polyphenol network coating; then, this coating is mixed with photosensitive liposomes encapsulating the first drug, multivalent metal ions, and a promoter in water to obtain the aforementioned photoresponsive drug delivery carrier system. The polyphenol is tannic acid, the multivalent metal ion is manganese ion Mn(II) derived from manganese chloride, and the promoter is 3-(N-morpholino)propanesulfonic acid.

[0105] Preferably, the mass-to-volume ratio of the photosensitive liposome loaded with the first drug to the intermediate product is 1 mg:(1-10) mL. Specifically, it is 1 mg:5 mL.

[0106] The fourth aspect of this invention protects the use of the photoresponsive drug delivery carrier or the photoresponsive drug delivery system described above in the preparation of antitumor drugs.

[0107] In some embodiments, the tumor is selected from one or more of melanoma, kidney cancer, liver cancer, thyroid cancer, gastric cancer, glioma, and breast cancer.

[0108] The present invention further provides a method for treating tumors, comprising administering an effective amount of a photoresponsive drug delivery system or an antitumor drug as described above to a desired subject. The method may also be in vitro or non-therapeutic, and the subject may be a mammal, such as, but not limited to, humans, primates, livestock, pets, laboratory test animals, or captured wild animals. The subject is preferably a primate. The subject is most preferably a human. The subject may be a patient with a tumor or an individual seeking tumor prevention. The nanoparticles or pharmaceutical composition may be administered to the subject before, during, or after tumor treatment.

[0109] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0110] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0111] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0112] For porphyrin-phospholipid reference, see Jonathan F. et al. Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents. Nature Nanotech. Materials, 10(4):324-332. (2011).

[0113] Dasatinib is abbreviated as DAS in the following embodiments of this application; Sunitinib is abbreviated as S in the following embodiments of this application; Phosphate buffer is abbreviated as PB; Phosphate buffer is abbreviated as PBS; Porphyrin-phospholipid is abbreviated as PoP; Dioleoyllecithin is abbreviated as DOPC; Distearate phosphatidylcholine is abbreviated as DSPC; Ethylenediaminetetraacetic acid is abbreviated as EDTA; 3-(N-morpholino)propanesulfonic acid is abbreviated as MOPS.

[0114] MPEG-PCL is an oligomer composed of methoxylated poly(ethylene glycol) and poly(ε-caprolactone) linked by dynamic covalent bonds.

[0115] A2058 melanoma cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. Human umbilical vein endothelial cells (HUVECs) were purchased from Lifeline Cell.

[0116] like Figure 4 This is a schematic diagram of the preparation method of the photoresponsive drug delivery system of the present invention, wherein Filmhydration sonication is a thin-film hydration method; Lipo is a liposome; Transmembrane Ammonium sulfate gradient is a transmembrane ammonium sulfate; TA is tannic acid; Mn 2+ Manganese chloride is used. S@Lipo is a photosensitive liposome encapsulating sunitinib; MDAS-NP is a metal polyphenol coordination polymer micelle loaded with dasatinib; MS@Lipo encapsulates sunitinib and simultaneously loads dasatinib to form a photoresponsive drug delivery system.

[0117] Example 1: Preparation and Characterization of Photoresponsive Drug Delivery Carriers

[0118] In Example 1, a light-responsive drug delivery carrier without any drug loading was prepared. This included the following:

[0119] 1.1 Obtaining photoresponsive drug delivery carriers

[0120] First, polymer micelles were prepared, then metal polyphenol-coordinated polymer micelles were prepared by combining them with metal salts and polyphenols. These micelles were then loaded onto the surface of photosensitive liposomes to form a photoresponsive drug delivery carrier. The details are as follows:

[0121] 1.1.1 Preparation of Polymer Micelles

[0122] Polymer micelles (NPs) were prepared using a nanoprecipitation method, including the following:

[0123] 1) Dissolution: Weigh 20 mg of MPEG-PCL (polymer) (Mw = 5000:5000) and dissolve it in 1 mL of N,N-dimethylformamide. Shake to completely dissolve the polymer to obtain the organic phase.

[0124] Prepare 0.5M phosphate buffer (PB) by preparing sodium dihydrogen phosphate solution (0.5M) and disodium hydrogen phosphate solution (0.5M) separately, mix the two and adjust the pH to 7.4, and store at 4℃ as the aqueous phase.

[0125] 2) Drop addition: Pipette 4 mL of 0.5 M phosphate buffer into a 7 mL conical bottom reaction flask. Add 1 mL of the organic phase (polymer and drug) from step 1) to the reaction flask at a uniform rate, with an interval of more than 5 seconds between each addition to ensure the formation of uniform emulsion droplets. After the addition is complete, stir at 1600 rpm for 4 h.

[0126] 3) Purification: After stirring in step 2), transfer the solution to a dialysis membrane (MWCO 3.5kDa, 4.5mm), place it in dialysate (1L, PB solution), and dialyze at 4℃ with a slow rotation speed (300rpm) for 24h. Change the dialysate 4 times during the process until the free drug and organic phase in the solution are completely removed.

[0127] 4) Concentration: Transfer the solution after dialysis in step 3) to an ultrafiltration tube (50 mL, 10 kDa), centrifuge at low speed (2500 × g) to concentrate the sample volume to only 0.5 mL, and obtain polymer micelles (NP).

[0128] 1.1.2 Obtaining Metal-Polyphenol Coordination Polymer Micelles (M-NP)

[0129] Take 1 mg of NP obtained in step 1.1.1, add 5 μL of tannic acid solution (24 mM), and vortex to mix for 1 min. Then add 5 μL of manganese chloride solution (24 mM) and 0.05 mL of 3-(N-morpholino)propanesulfonic acid (MOPS) (100 mM, pH = 8.0) buffer, and vortex to mix for 1 min. After centrifuging the product (8000×g, 10 min), collect the supernatant, centrifuge and ultrafilter for 20 min (3500 g), and wash repeatedly 3 times to obtain the metal polyphenol coordination polymer micelles NP (M-NP).

[0130] 1.1.3 Obtaining photosensitive liposomes (Lipo)

[0131] Prepared by thin-film hydration method, including the following steps:

[0132] In a flask, a total of 40 mg of lipids (DOPC, DSPC, Cholesterol, PoP = 10.75:37.25:50:2 molar ratio) were dissolved in a mixed solution of methanol and chloroform (volume ratio 1:3), and the solution was evaporated in a vacuum rotary evaporator at 60 °C for 1 h. The mouth of the round-bottom flask was then plugged, leaving a certain amount of vent, and the flask was placed in a vacuum drying oven for overnight drying.

[0133] The following day, remove the round-bottom flask and add 4 mL of a mixed solution of ammonium sulfate and EDTA (the final concentration of EDTA in the mixed solution is 4 wt%, and the concentration of ammonium sulfate is 250 mM) for hydration. After hydration, the solution is repeatedly extruded 10 times through a liposome extruder (200 nm), and dialyzed overnight at 4°C to remove free ammonium sulfate. The resulting photosensitive liposomes (Lipo) are then obtained.

[0134] 1.1.4. Obtaining a photoresponsive drug delivery carrier (Lipo)

[0135] Mix 1 mg of M-NP from step 1.1.2 with 0.5 mg of Lipo from step 1.1.3 thoroughly, add 5 μL of manganese chloride solution (24 mM), and vortex for 30 s. Then add 0.05 mL of MOPS (100 mM, pH 8.0) buffer and incubate with vortexing for 30 s. Centrifuge the solution at 3000 g for 30 min to obtain the successfully assembled photoresponsive drug delivery carrier (M-NP-Lipo), with the structure shown below. Figure 1 As shown.

[0136] Figure 1 In this study, the surface of polymer micelles (NP) is coated with a metal polyphenol network coating (M), and the surface of photosensitive liposomes (Lipo) is loaded with metal polyphenol coordination polymer micelles (M-NP).

[0137] 1.2 FRET was used to investigate the assembly stability of the photoresponsive drug delivery carrier.

[0138] To confirm the successful assembly of the photoresponsive drug delivery carrier, a fluorescent probe DiO (FRET donor) was used to label the polymer micelles NP, and a fluorescent probe DiI (FRET acceptor) was used to label the photosensitive liposome Lipo bilayer.

[0139] DAS-NP(DiO) group: DiO is labeled in polymer micelles NP.

[0140] Lipo(DiI) group: DiI is labeled in photosensitive liposomes Lipo.

[0141] DAS-NP(DiO)+Lipo(DiI) group: The above DAS-NP(DiO) group and Lipo(DiI) group are physically mixed at a volume ratio of 2:1.

[0142] MS@Lipo(DiO+DiI) group: DiO was labeled on polymer micelles NP and DiI was labeled on photosensitive liposomes Lipo, and photoresponsive drug delivery carriers labeled with DiO and DiI were prepared according to the same method as step 1.1 in Example 1.

[0143] Take 200L of the final product from each group and add it to a black-bottomed 96-well plate. Place the plate in a microplate reader to detect fluorescence (Ex = 405nm, Em = 410nm-600nm).

[0144] See results Figure 2 .

[0145] from Figure 2 It can be seen that the fluorescence intensity of DiO at 500 nm is weakened in the MS@Lipo(DiO+DiI) group, while the FRET signal at 565 nm is enhanced. This indicates that the photosensitive liposomes are connected with the metal polyphenol coordination polymer micelles to form a core-satellite structure with the photosensitive liposome Lipo as the core and the metal polyphenol coordination polymer micelles M-NP as the satellites.

[0146] from Figure 2 It is known that DAS-NP(DiO) exhibits its maximum emission peak (520nm) when excited at an Ex of 405nm. Lipo(DiI) shows no emission peak when excited at an Ex of 405nm, proving that 405nm cannot excite DiI to produce a fluorescence signal. Physical mixing of DAS-NP(DiO) and Lipo(DiI) (i.e., the DAS-NP(DiO) + Lipo(DiI) group) only produces a fluorescence signal at 515nm, meaning that only the DiO signal is observed after physical mixing. However, when DAS-NP(DiO) and Lipo(DiI) are assembled into a photoresponsive drug delivery carrier (core-satellite structure), not only is the laser emission peak of DiO observed at 405nm, but the maximum fluorescence signal of DiI at 570nm can also be observed. This fully demonstrates that the physical distance between the polymer micelles and the photosensitive liposomes is sufficiently close, and the photosensitive liposomes are connected to the polymer micelles, thus forming a core-satellite system with the photosensitive liposome as the core and the metal polyphenol-coordinated polymer micelles as the satellites.

[0147] 1.3 Investigation into the deep penetration of photoresponsive drug delivery carriers into tumor spheres

[0148] The study investigated the deep penetration of photoresponsive drug delivery carriers into tumor spheres. This included the following:

[0149] In vivo tumors present a heterogeneous environment, with deep hypoxia in solid tumors. This study investigates the deep penetration ability of photoresponsive drug delivery carriers within tumor spheres using a tumor sphere experiment, thereby simulating the uptake behavior of photoresponsive drug delivery carriers in tumor tissue in vitro. Tumor cells are cultured in low-absorption plates to form dense tumor spheres, with hypoxic tumor cells inside and normally proliferating tumor cells on the outside.

[0150] Tumor spheroid culture: A2058 cells were cultured at a concentration of 2.5 × 10⁻⁶. 4 Inoculate cells at a density of 1 cell / mL (in DMEM complete medium) into low-adsorption 96-well round-bottom plates. Immediately centrifuge the plates at 1000g for 10 min at 4°C. Incubate for 24-72 h until cells form uniform spherical structures.

[0151] To observe the penetration of photoresponsive drug delivery carriers into deep tumors, tumor spheres were used as a simulation. Coumarin 6 was loaded into MPEG-PCL polymer micelles (the loading method was the same as in Example 2, 2.1.1, except that dasatinib was replaced with coumarin 6). Then, a metal polyphenol network coating was encapsulated using the method described in Step 1.1.2 of Example 1, and combined with the photosensitive liposomes (Lipo) from Step 1.1.3 of Example 1 to form a coumarin-labeled photoresponsive drug delivery carrier (labeled coumarin 6@Lipo, which was unloaded with any drug). The experiment was conducted in two groups:

[0152] ①Coumarin 6@Lipo-Light group: Coumarin 6@Lipo unlit group;

[0153] ②Coumarin 6@Lipo+Light group:Coumarin 6@Lipo at 660nm, 300mW / cm 2 Irradiate for 5 minutes.

[0154] Two groups of pretreated coumarin 6@Lipo were added to A2058 tumor spheres that had been cultured into spherical shapes and incubated for 3 hours. The supernatant culture medium was discarded, and the tumor spheres were carefully transferred to glass dishes and placed under a confocal microscope (CLSM) to observe the fluorescence changes in the tumor spheres. The fluorescence intensity of the cells after the Z-axis changes can be photographed to visually show the penetration of coumarin 6@Lipo into the deep part of the tumor.

[0155] See results Figure 3 .

[0156] from Figure 3As shown in Figure A, after incubating A2058 tumor spheres with either light-treated or untreated coumarin 6@Lipo, the fluorescence of coumarin 6 at different tumor depths along the Z-axis was observed using CLSM. The results showed that clear red fluorescence signals of coumarin 6 could be observed not only at the periphery of the tumor spheres but also inside them. Significant fluorescence signals were observed within 100-300 μm of the tumor spheres in the light-treated group (coumarin 6@Lipo+Light); while in the untreated group (coumarin 6@Lipo-Light), coumarin 6 fluorescence was only observed in the outermost cells of the tumor spheres.

[0157] from Figure 3 As shown in Figure B, statistical analysis of the fluorescence signal at 300 μm revealed that the coumarin 6@Lipo+Light group exhibited a significant fluorescence signal, while the coumarin 6@Lipo-Light group only showed strong fluorescence signals at the lateral boundaries of the tumor spheres, with no obvious fluorescence signal inside. This demonstrates that after near-infrared light induces the disintegration of the nuclear satellite structure, small-diameter polymer micelles have the ability to penetrate deep into the tumor.

[0158] The results show that polymer micelles in the light-pretreated group can penetrate deep into the tumor sphere, and also indicate the potential for nuclear satellite structure disintegration and sequential drug delivery of the drug delivery carrier under light response.

[0159] Example 2: Acquisition and Characterization of a Photoresponsive Drug Delivery System

[0160] according to Figure 4 The flowchart shown illustrates the preparation and characterization of a drug-loaded, photoresponsive drug delivery system (MS@Lipo).

[0161] Including the following:

[0162] 2.1 Construction of a photoresponsive drug delivery system (MS@Lipo)

[0163] 2.1.1 Preparation of polymer micelles loaded with dasatinib

[0164] Small-particle-size polymer micelles (DAS-NP) loaded with a second drug were prepared using a nanoprecipitation method, including the following:

[0165] 1) Dissolution: Weigh 0.6 mg dasatinib (second drug) and 20 mg MPEG-PCL (polymer) (Mw = 5000:5000) and dissolve them in 1 mL N,N-dimethylformamide. Shake to dissolve completely to obtain the organic phase.

[0166] Prepare 0.5M phosphate buffer (PB) by preparing sodium dihydrogen phosphate solution (0.5M) and disodium hydrogen phosphate solution (0.5M) separately, mix the two and adjust the pH to 7.4, and store at 4℃ as the aqueous phase.

[0167] 2) Drop addition: Pipette 4 mL of 0.5 M phosphate buffer into a 7 mL conical bottom reaction flask. Add 1 mL of the organic phase (polymer and drug) from step 1) to the reaction flask at a uniform rate, with an interval of more than 5 seconds between each addition to ensure the formation of uniform emulsion droplets. After the addition is complete, stir at 1600 rpm for 4 h.

[0168] 3) Purification: After stirring in step 2), transfer the solution to a dialysis membrane (MWCO 3.5kDa, 4.5mm), place it in dialysate (1L, PB solution), and dialyze at 4℃ with a slow rotation speed (300rpm) for 24h. Change the dialysate 4 times during the process until the free drug and organic phase in the solution are completely removed.

[0169] 4) Concentration: Transfer the solution after dialysis in step 3) to an ultrafiltration tube (50 mL, 10 kDa), centrifuge at low speed (2500 × g) to concentrate the sample volume to only 0.5 mL, and obtain small-particle-size drug-loaded polymer micelles (DAS-NP).

[0170] 2.1.2 Obtaining metal polyphenol coordination polymer micelles (MDAS-NP) loaded with dasatinib

[0171] Take 1 mg of DAS-NP obtained in step 2.1.1, add 5 μL of tannic acid solution (24 mM), and vortex to mix for 1 min. Then add 5 μL of manganese chloride solution (24 mM) and 0.05 mL of 3-(N-morpholino)propanesulfonic acid (MOPS) (100 mM, pH = 8.0) buffer, and vortex to mix for 1 min. After centrifuging the product (8000×g, 10 min), collect the supernatant, centrifuge and ultrafilter for 20 min (3500 g), and wash repeatedly 3 times to obtain dasatinib-loaded metal polyphenol coordination polymer micelles DAS-NP (MDAS-NP).

[0172] 2.1.3 Obtaining liposomes (S@Lipo) encapsulating sunitinib

[0173] Prepared by thin-film hydration method, including the following steps:

[0174] In a flask, a total of 40 mg of lipids (DOPC, DSPC, Cholesterol, PoP = 10.75:37.25:50:2 molar ratio) were dissolved in a mixed solution of methanol and chloroform (volume ratio 1:3), and the solution was evaporated in a vacuum rotary evaporator at 60 °C for 1 h. The mouth of the round-bottom flask was then plugged, leaving a certain amount of vent, and the flask was placed in a vacuum drying oven for overnight drying.

[0175] The next day, remove the round-bottom flask and add 4 mL of a mixed solution of ammonium sulfate and EDTA (the final concentration of EDTA in the mixed solution is 4 wt%, and the concentration of ammonium sulfate is 250 mM) for hydration. After hydration, the solution is repeatedly extruded 10 times through a liposome extruder (200 nm), and dialyzed overnight at 4 °C to remove free ammonium sulfate.

[0176] Sunitinib (the first-line drug) was added at a drug-to-liposome ratio of 1:8, and the mixture was shaken on a constant-temperature shaker for 1 hour (60°C, 250 rpm). The drug-to-liposome ratio refers to the mass ratio of drug molecules to lipid molecules in the liposomes.

[0177] Free sunitinib not encapsulated in photosensitive liposomes can be obtained by removing the free sunitinib by ultrafiltration (2000 rpm, 10 kDa).

[0178] 2.1.4 Acquisition of the photoresponsive drug delivery system (MS@Lipo)

[0179] Mix 1 mg of MDAS-NP from step 2.1.2 with 0.5 mg of S@Lipo from step 2.1.3 until homogeneous, add 5 μL of manganese chloride solution (24 mM), and vortex for 30 s. Then add 0.05 mL of MOPS (100 mM, pH = 8.0) buffer and vortex for 30 s. Centrifuge the solution at 3000 g for 30 min to obtain the successfully assembled photoresponsive drug delivery system (MS@Lipo).

[0180] Meanwhile, the metal polyphenol coordination polymer micelles DAS-NP loaded with dasatinib from step 2.1.1 and the photosensitive liposomes without sunitinib were prepared in the same way to obtain a photoresponsive drug delivery system loaded only with dasatinib (labeled M@Lipo).

[0181] Meanwhile, the photosensitive lipid S@Lipo loaded with sunitinib from step 2.1.3 and the metal polyphenol coordination polymer micelles without dasatinib were prepared in the same way to obtain a photoresponsive drug delivery system loaded only with sunitinib (labeled S@Lipo).

[0182] Example 3 Characterization of the photoresponsive drug delivery system (MS@Lipo)

[0183] In Example 3, the polymer micelles loaded with dasatinib (DAS-NP), the metal polyphenol coordination polymer micelles loaded with dasatinib (MDAS-NP), the photosensitive liposomes encapsulating sunitinib (S@Lipo), and the photoresponsive drug delivery system simultaneously encapsulating dasatinib and loading sunitinib (MS@Lipo) prepared in Example 2 were characterized. The characteristics included the following:

[0184] 3.1 Investigation of particle size, potential, encapsulation efficiency, and drug loading

[0185] 1) Particle size, potential

[0186] Particle size and potential were detected using dynamic light scattering (DLS), specifically measured using a Zetasizer NanoZS laser particle size analyzer. For direct measurement of particle size, 100L of sample was placed in a dedicated particle size measurement dish. For potential measurement, 20L of sample was thoroughly dispersed in 1mL of triple-distilled water, mixed evenly, and then placed in a dedicated potential measurement dish.

[0187] 2) Determination of the encapsulation efficiency of sunitinib

[0188] Preparation of standard solutions: Weigh sunitinib standard and dissolve it in triple-distilled water to prepare a 1 mg / mL standard solution. Serially dilute with chromatographic grade methanol to obtain standard solutions of various concentrations: 2, 5, 10, 20, 50, 100, 150, and 200 μg / mL. Store at 4℃.

[0189] Sample preparation: S@Lipo and MS@Lipo were added to methanol and vortexed for 0.5 h to fully react and disrupt the liposome bilayer membrane. The supernatant was then collected by centrifugation at 14800 rpm for 10 min.

[0190] HPLC detection: Samples were separated using a Diamonsil C18 column (150 × 4.6 mm). ① Mobile phase: methanol, water, acetonitrile (18:52:30, V / V / V), 0.1% trifluoroacetic acid, sonicated for 10 min to remove air bubbles. ② Experimental conditions: flow rate 1.0 mL / min, UV detection wavelength 420 nm, injection volume 10 μL.

[0191] Drug content determination: All solutions were added to the injection vial. Standard solutions were injected into the syringe from low to high concentrations, followed by washing the syringe with blank solvent, and then the sample was injected. The actual concentration of sunitinib in the sample was calculated using the peak area curve.

[0192] Encapsulation efficiency (EE) is the ratio of the actual amount of drug contained in a unit weight of a photoresponsive drug delivery system (S@Lipo or MS@Lipo) to the original dosage. The calculation method is as follows:

[0193] Encapsulation rate (%) = Actual drug mass / Drug dosage × 100%

[0194] 3) Assay for the drug loading of dasatinib

[0195] Preparation of standard solutions: Weigh dasatinib standard and dissolve it in chromatographic grade methanol to prepare a 1 mg / mL standard solution. Then, use methanol to serially dilute the solution to prepare standard solutions of different concentrations: 2, 5, 10, 20, 50, 100, 200, and 500 μg / mL. Store at 4℃ for later use.

[0196] Sample preparation: DAS-NP, MDAS-NP, and MS@Lipo were added to dimethyl sulfoxide and vortexed for 0.5 h to fully react and destroy the polymer micelle structure. Then, the dimethyl sulfoxide was removed by vacuum concentrator. The sample was redissolved in 400 μL of chromatographic grade methanol, centrifuged at 14800 rpm for 10 min, and the supernatant was collected. 200 μL of the supernatant was added to a vial.

[0197] HPLC detection: ① Mobile phase: water, methanol, acetonitrile (65:5:30, V / V / V), containing 0.1% formic acid. Sonicate for 10 min to remove air bubbles. ② Experimental conditions: flow rate 1.0 mL / min, UV detection wavelength 320 nm, injection volume 10 μL.

[0198] Drug content determination: All solutions were added to the injection vial. Standard solutions were injected into the syringe from low to high concentrations, followed by washing the syringe with blank solvent, and then the sample was injected. The actual concentration of dasatinib in the sample was calculated using the peak area curve.

[0199] Drug loading (DL) refers to the total mass of drug contained in a unit weight of nanoparticles. Nanoparticles refer to DAS-NP, MDAS-NP, and MS@Lipo. The calculation method is as follows:

[0200] Drug loading (%) = (Actual drug mass loaded in nanoparticles / Total mass of drug-loaded nanoparticles) × 100%

[0201] The results are shown in Table 1.

[0202] Table 1

[0203]

[0204] As shown in Table 1, compared with DAS-NP, the particle size of MDAS-NP is slightly increased to 48 nm, the Zeta potential is smaller, and the drug loading is not significantly changed. When MDAS-NP is attached to the S@Lipo surface, the particle size of the resulting MS@Lipo continues to increase to 182 nm, and the surface is negatively charged.

[0205] As shown in Table 1, DLS analysis revealed that the particle size distributions of DAS-NP, MDAS-NP, S@Lipo, and MS@Lipo were all unimodal and the PDI values ​​were all less than 0.2, indicating that the particle distribution within the system was uniform, the preparation process was stable, and the photoresponsive, sequential drug delivery system was successfully prepared.

[0206] 3.3 Photoresponsive characteristics of the photoresponsive drug delivery system (MS@Lipo)

[0207] The chelation of phenolic hydroxyl groups with metal ions in polyphenols is a reversible reaction, influenced by competing metal chelates and pH. The metal-polyphenol network structure is more robust in alkaline environments but becomes looser under acidic conditions, as the coordination bonds in the polyphenol network easily break. When a stronger metal chelating agent is present in the solution, the original metal ions on the metal-polyphenol network are replaced by the new chelating agent, forming new chelated products. Therefore, to study the photoresponse characteristics of MS@Lipo, the chelation properties of polyphenols with a stronger iron chelating agent were utilized to detect the release characteristics of EDTA under photoresponse.

[0208] Three treatment groups were set up, with 1 mL of MS@Lipo taken from each group and subjected to different treatments: ① Untreated group (-light); ② 10% Triton treatment group (+Triton): MS@Lipo was added to a final concentration of 10% Triton and mixed for 30 min; ③ Light treatment group (+light): The light conditions were 660 nm and 300 mW / cm². 2 , 5 minutes.

[0209] The solutions from each group were transferred to a 10 kDa ultrafiltration tube and ultrafiltered (3000 g, 0.5 h). The filtrate was collected, and an appropriate amount of sulfuric acid was added to each group to adjust the solution environment to 0.1 NH₂SO₄. Subsequently, excess Fe was added. 3+ After the solution is mixed evenly, it is added to a quartz dish and placed in a Nanodrop instrument to detect the ultraviolet absorption peak at 258 nm of the sample.

[0210] At the same time, different concentrations of free EDTA and Fe were set. 3+ The standard curve of the reaction was obtained for different concentrations of free EDTA, namely 0.1 mM, 0.3 mM, 0.5 mM, 1 mM, 3 mM, 5 mM and 10 mM.

[0211] Based on the standard curve, the EDTA content in each group of solutions can be calculated, thereby revealing the EDTA release in the photoresponsive phase.

[0212] like Figure 5 As shown. A) Different concentrations of free EDTA and Fe 3+(A) UV-Vis absorption spectrum of the reaction, showing a characteristic absorption peak at 258 nm. (B) Release of EDTA from MS@Lipo after light irradiation. (C) Nanosight detection of particle size changes in MS@Lipo before and after light irradiation. (D) DLS detection of particle size changes in MS@Lipo before and after light irradiation.

[0213] from Figure 5 As shown in A, iron ions and free EDTA have a characteristic absorption peak at 258 nm.

[0214] from Figure 5 As shown in Figure B, the untreated group (-light) had no characteristic absorption peak at 258 nm; the light-treated group (+light) had a distinct characteristic absorption peak at 258 nm; and the 10% Triton-treated group (+Triton) had a distinct characteristic absorption peak at 258 nm. The intensity of the characteristic absorption peaks that disrupted the liposome membrane was the same in the light-treated group and the 10% Triton-treated group, proving that light irradiation can cause the complete release of EDTA in the photoresponsive drug delivery system, which in turn can promote the disintegration of the structure of the liposome core-polymer micelle satellite.

[0215] from Figure 5 According to Nanosight, after light treatment, MS@Lipo changed from a uniform, relatively large particle size (approximately 200 nm) before light exposure to a smaller particle size (approximately 40 nm). The 40 nm signal is precisely the particle size of the polymer micelles DAS-NP loaded with dasatinib.

[0216] from Figure 5 According to DLS, the same particle size change was observed, that is, after light irradiation, the particle size changed from about 200 nm to about 40 nm, indicating that the photosensitive liposomes disintegrated after light irradiation, leaving polymer micelles.

[0217] Meanwhile, a photoresponsive drug delivery system was prepared using the same method as in Example 2, consisting of photosensitive liposomes without EDTA in the inner water cavity but encapsulating sunitinib and metal polyphenol coordination polymer micelles without a second drug loading. The particle size changes before and after light exposure were then observed. Figure 6 .

[0218] from Figure 6 It can be seen that the particle size of the photoresponsive drug delivery system did not change significantly before and after light exposure, and the particle size was still about 200 nm after light exposure, indicating that the polymer micelles could not detach from the surface of the photosensitive liposomes.

[0219] The above results fully demonstrate that MS@Lipo containing EDTA in its internal water cavity has photoresponsive structural disintegration capability, enabling rapid in vivo release of sunitinib from the photosensitive liposome.

[0220] 3.4. Photoresponsive drug delivery system (MS@Lipo) photoresponsive drug release capability

[0221] To further investigate the effect of light irradiation on drug release, the MS@Lipo samples were divided into two groups:

[0222] 1) No light group (-light): MS@Lipo without light exposure;

[0223] 2) Illumination treatment group (+light): MS@Lipo illumination conditions were 660nm, 300mW / cm². 2 , 5 minutes.

[0224] Both groups of samples were placed in a constant-temperature shaker (37℃) and shaken at 200 rpm for 48 h. At 0, 1, 2, 4, 8, 12, and 24 h, 1 mL of solution was taken and purified by ultrafiltration, collecting the filtrate (3000 g, molecular weight cutoff MWCO of 3 kDa). The filtrate was treated with methanol and then evaporated to dryness in a vacuum concentrator. It was then reconstituted in methanol, and the contents of dasatinib and sunitinib were determined by HPLC. The detection conditions are described in step 3.2, and the drug content was calculated based on the peak areas of the standards and samples.

[0225] like Figure 7 As shown in the figure, A represents the release rate of dasatinib from MS@Lipo within 24 hours with and without light treatment; B represents the release rate of sunitinib from MS@Lipo within 24 hours with and without light treatment, and the release rate of sunitinib from MS@Lipo within 10 minutes after light treatment, respectively. Here, -light indicates no light treatment, +light indicates light treatment; DAS release represents the release rate of dasatinib, and Sunitinib release represents the release rate of sunitinib.

[0226] from Figure 7 As shown in the data, without light treatment, the release of dasatinib within 24 hours is less than 10%, while after light treatment, the release of dasatinib can be increased to nearly 40% within 12 hours; indicating that the release of dasatinib is significantly enhanced after light irradiation.

[0227] from Figure 7 As is known from the data, without light treatment, the release rate of sunitinib within 24 hours is less than 10%, while after light treatment, sunitinib exhibits high photoresponsiveness, with a release rate of 100% within 5 minutes.

[0228] The above results fully demonstrate that the MS@Lipo photoresponsive drug delivery system has excellent photoresponsive drug release capability.

[0229] The good stability of photoresponsive drug delivery systems in the bloodstream is fundamental to the anti-tumor effects of nanomedicines. About half of the plasma proteins in blood are albumin, which has a high drug-binding capacity. When drug-encapsulated nanocarriers move under shear stress in narrow capillaries, these proteins may affect the stability of the nanoparticles.

[0230] In vitro simulation of in vivo blood circulation conditions was performed by placing the MS@Lipo drug delivery system in PBS containing 10% FBS and shaking at 37°C for 24 hours. DLS showed that the particle size of the drug delivery system remained basically stable and the PDI did not increase significantly, proving that the drug delivery system did not experience large-scale aggregation or disintegration and has good blood circulation stability.

[0231] In addition, the long-term storage stability of the drug delivery system is also crucial. DLS observation showed that the particle size and PDI of MS@Lipo did not change significantly after 7 days of storage in PBS (pH 7.4, 4℃), indicating that the preparation process of the drug delivery system is stable and has good storage stability.

[0232] Example 4: Investigation of HUVEC Cell Migration and Tubule Formation Using the Photoresponsive Drug Delivery System S@Lipo. In Example 4, the photoresponsive drug delivery system S@Lipo obtained in Example 2 was used to study the migration and tubule formation of human umbilical vein endothelial cells (HUVECs) in vitro, to verify the anti-angiogenic effect of S@Lipo (where the polymer micelles in the nucleus-satellite structure do not load dasatinib, but only sunitinib is encapsulated in photosensitive liposomes). This includes the following:

[0233] 4.1 HUVEC cell experimental grouping

[0234] The experimental group consists of:

[0235] 1) S@Lipo group: S@Lipo was not subjected to light treatment;

[0236] 2) S@Lipo+L group: S@Lipo at 660nm, 300mW / cm 2 Light pretreatment for 5 minutes.

[0237] The concentrations of sunitinib in the S@Lipo group and the S@Lipo+L group were 5, 10, and 20 μM, respectively.

[0238] 4.2 Investigation of HUVEC cell migration and tubule formation

[0239] Angiogenesis is a crucial source of nutrients for tumor cells to absorb and metastasize. To better eradicate tumor cells, in addition to inhibiting tumor cell activity, it is necessary to block tumor angiogenesis, thereby depriving tumor cells of nutrients and preventing further development. Cell migration and tubule formation can simulate the effects of angiogenesis within tumors in vitro. Tubular formation assays are a rapid quantitative method for measuring in vitro angiogenesis.

[0240] 4.2.1 HUVEC cell migration

[0241] HUVEC cells (purchased from Lifeline Cell) were kept at a density of 4 × 10⁻⁶. 5 When the cell / mL ratio was 100 cells / mL, the experiment was performed using a Transwell chamber. 700 μL of complete HUVEC culture medium was added to the lower layer of the chamber, and 100 μL of digested HUVEC cells was added to the upper chamber. The light-responsive drug delivery system for each group was then added to the upper chamber.

[0242] A negative control group (Control) was set up without any treatment, i.e., no nanoparticles were added, only HUVEC complete medium was added. Each group was set up with 3 replicates as parallel controls.

[0243] The Transwell chambers were then incubated at 37°C for 8 hours. The plates were then removed, and the culture medium in both the upper and lower chambers was discarded. 600 μL of 4% paraformaldehyde was added to fix the cells for 15 minutes. The cells were washed 2-3 times with DPBS, and any unmigrated cells in the upper chamber were gently wiped away with a fine cotton swab. 100 μL of 0.1% crystal violet was added, and the cells were stained overnight. The cells were then washed 3 times with DPBS until no excess crystal violet remained. Images were taken at 100x magnification using a normally upright microscope. Cell migration rates were calculated for each group.

[0244] Cell migration rate (%) = (Number of cells in the experimental group / Number of cells in the negative control group) × 100%

[0245] 4.2.2 HUVEC cell tubule formation

[0246] 24 hours in advance, freeze 96-well plates and sterilized pipette tip boxes (200 μL) at -20°C. Thaw the matrix gel overnight at 4°C, allowing it to change from a solid to a liquid state. Place an ice plate in a clean bench beforehand, and then place the pre-treated matrix gel, 96-well plates, and pipette tip boxes on its surface. Pipette 70 μL of matrix gel onto the surface of the 96-well plate, handling this step slowly to avoid air bubbles or uneven surfaces. Let the 96-well plates with the matrix gel added stand on the ice plate for 10 minutes, then transfer them to a 37°C cell culture incubator and incubate for 30 minutes until the matrix gel solidifies. After the gel is laid, digest and disperse HUVECs to a cell density of 1 × 10⁻⁶ cells / well. 5Single-cell suspensions of 10 cells / mL were added to 96-well plates after gel coating, along with the aforementioned photoresponsive drug delivery systems. The plates were then incubated at 37°C for 10 hours. Tube formation was observed in each group, and the tube formation inhibition rate was calculated.

[0247] The formula for calculating the tubule formation inhibition rate is as follows:

[0248] Tubuloformation inhibition rate (%) = (Tubuloform length of negative control group - Tubuloform length of experimental group) / Tubuloform length of negative control group × 100%

[0249] 4.3 Results of HUVEC cell observation

[0250] 4.3.1 Inhibition of HUVEC cell migration

[0251] like Figure 8 As shown. Representative photographs of crystal violet staining of migrating HUVECs after co-incubation with S@Lipo (10 μM sunitinib) pretreated with light; and statistical graphs of the migration rate of HUVECs in S@Lipo with different sunitinib concentrations under light pretreatment or no light treatment.

[0252] from Figure 8 The crystal violet staining experiment showed that light pretreatment of S@Lipo (S@Lipo+L) more effectively inhibited HUVEC migration. The inhibition of HUVEC migration increased with the concentration of sunitinib in S@Lipo (5, 10, 20 μM), and the light pretreatment group containing 20 μM sunitinib almost completely inhibited HUVEC migration, with a migration rate of less than 5%.

[0253] This demonstrates that the S@Lipo photoresponsive drug delivery system can release sunitinib from the water cavity of photosensitive liposomes under near-infrared light response, thereby exerting the anti-angiogenic activity of sunitinib.

[0254] 4.3.2 Inhibition of HUVEC cell tubule formation

[0255] like Figure 9 As shown in the figure, A represents a representative photograph of HUVEC tubule formation after incubation with HUVEC in the S@Lipo group without light pretreatment (containing 5, 10, and 20 μM sunitinib, respectively); B represents a representative photograph of HUVEC tubule formation after incubation with HUVEC in the S@Lipo+L group with light pretreatment (containing 5, 10, and 20 μM sunitinib, respectively); C represents a representative photograph of HUVEC tubule formation in the negative control group; and D represents the tubule formation inhibition rate of S@Lipo with different sunitinib concentrations (5, 10, and 20 μM) with or without light pretreatment.

[0256] from Figure 9 As shown in Figure D, the ability of HUVEC tubules to form was gradually inhibited as the concentration of sunitinib in the S@Lipo group and the S@Lipo+L group increased (5, 10, 20 μM), and the inhibition effect was better in the S@Lipo+L group after light pretreatment.

[0257] from Figure 9 As shown in A, B, and C, the S@Lipo+L group under light pretreatment completely inhibited tubule formation at the lowest concentration of 5 μM, and tubule branching and tubular network formation as shown in the negative control group (Control) could not be observed.

[0258] Example 5: Investigation of A2058 Cell Migration and Tubule Formation Using the Photoresponsive Drug Delivery System M@Lipo. In Example 5, the photoresponsive drug delivery system M@Lipo obtained in Example 2 was used to study the migration and tubule formation of A2058 cells in vitro, to verify the anti-angiogenic effect of M@Lipo (where the photosensitive liposomes in the nucleus-satellite structure do not encapsulate sunitinib, but only polymer micelles load dasatinib). This included the following:

[0259] 5.1 A2058 cell experimental grouping

[0260] The experiment was divided into two groups:

[0261] 1) M@Lipo-Light group: M@Lipo is not exposed to light.

[0262] 2) M@Lipo+Light group: M@Lipo at 660nm, 300mW / cm 2 Irradiation pretreatment for 5 minutes.

[0263] In both the M@Lipo-Light and M@Lipo+Light groups, four concentration gradients of dasatinib were set: 10, 30, 100, and 300 nM.

[0264] 5.2 Investigation of A2058 cell migration and tubule formation

[0265] 5.2.1 A2058 cell migration

[0266] A2058 cells were used at a rate of 1×10 4Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured overnight to allow cell adhesion. The medium was replaced with fresh medium, and cells were cultured until confluence reached 90%. The medium was then discarded, and the cells were washed once with DPBS. Using the IncuCyte automated cell scratching tool, uniformly wide cell wounds were made on the bottom of the 96-well plate in a single stroke. The cells were then washed once with DPBS, serum-free medium was added, and the plate was placed in an IncuCyte incubator where cell migration was automatically monitored over 24 hours.

[0267] A negative control (Control) was set up without any treatment, i.e., without the addition of any nanoparticles, using only the culture medium for culturing A2058 cells. Each group was set up with 3 replicates as parallel controls.

[0268] 5.2.2 Tubule Formation in A2058 Cells

[0269] A2058 cells were dispersed at a density of 1×10⁻⁶. 5 Single-cell suspensions of cells / mL were added to the pretreated M@Lipo light-responsive drug delivery system for each group. After thorough mixing, the mixture was added to 96-well plates that had been coated with gel and placed in a 37°C cell culture incubator. After 10 hours of culture, the 96-well plates were removed, washed once with DPBS, and observed under an inverted optical microscope to assess cell state and tubule formation. Tubule formation images were taken for each group, with three replicates for each experiment. Tubule formation was observed in each group, and the tubule formation inhibition rate was calculated. The inhibition rate was calculated as in section 4.2.2.

[0270] A negative control group (Control) was set up without any treatment, i.e., without the addition of any nanoparticles, using only the culture medium for culturing A2058 cells. Each group was set up with 3 replicates as parallel controls.

[0271] 5.3 Results of A2058 cell examination

[0272] 5.3.1 A2058 cell migration

[0273] like Figure 10 As shown in Figure A. A represents the effect of M@Lipo with or without light treatment on the migration ability of A2058 cells observed by IncuCyte. Dasatinib concentrations were 10, 30, 100, and 300 nM. B represents the quantitative analysis of cell healing ability in Figure A.

[0274] from Figure 10 As shown in Figure A, the cell scratch assay revealed that, compared with the scratch healing area of ​​the negative control group, the scratch healing ability of the photoresponsive drug delivery system M@Lipo, whether pretreated with light or not, decreased with increasing dasatinib concentration, and the two were negatively correlated.

[0275] from Figure 10 As shown in Figure B, the near-infrared light-treated M@Lipo+Light group showed stronger inhibition of scratch healing ability than the untreated M@Lipo-Light group. This indicates that near-infrared light effectively promotes the release of dasatinib from the polymer micelles of the nucleus-satellite structure, thereby increasing the amount of dasatinib released and enabling it to exert its ability to inhibit the migration of A2058 melanoma cells.

[0276] 5.3.2 Tubule Formation in A2058 Cells

[0277] like Figure 11 As shown. Representative photographs of A2058 tubule formation and quantitative results of tubule formation inhibition rate after co-incubation of M@Lipo (dasatinib: 10, 30, 100, 300 nM) with A2058 after light pretreatment or no light treatment.

[0278] from Figure 11 It was found that, compared with the untreated M@Lipo-Light group, when A2058 cells were incubated with the light-pretreated M@Lipo+Light group, the length, number of loops, and number of nodes in the A2058 cells were significantly reduced, and the tubule formation ability was significantly inhibited. The inhibitory effect increased with increasing dasatinib concentration, and the two were positively correlated with the dose. When the dasatinib concentration reached 300 nM, tubule formation in the light-pretreated or untreated M@Lipo groups was completely inhibited, with a value of 0.

[0279] The results demonstrate that the polymer micelles loaded with dasatinib in the M@Lipo photoresponsive drug delivery system can respond to near-infrared light to achieve structural disintegration and release of dasatinib, exerting a significant anti-VM effect in A2058 cells.

[0280] Example 6: In vivo tumor killing effect of the photoresponsive drug delivery system (MS@Lipo)

[0281] In this Example 6, the photoresponsive drug delivery system (MS@Lipo) obtained in Example 2 was used to investigate tumor killing in vivo, including the following:

[0282] 6.1 Construction of Melanoma Model

[0283] 3-4 week old BALB / c nude mice were housed in a barrier environment for one week to acclimatize before being inoculated with tumor cells. 100 μL of cells were aspirated from the insulin syringe at a cell density of 2 × 10⁻⁶. 7 A single-cell suspension of A2058 cells / mL was injected subcutaneously into the right back of the mouse. The needle was advanced slowly until a cell bulge formed under the skin. The insulin needle was then slowly withdrawn to prevent leakage of the cell suspension. The mouse was returned to its cage. Due to the slow growth cycle of human-derived cell tumors, tumor formation was observed at the injection site after three weeks.

[0284] The preparation method for A2058 single-cell suspension is as follows: Take A2058 tumor cells, discard the original culture medium, and rinse the culture flask twice with 5 mL L PBS to ensure no culture medium residue remains. Add 2 mL of 0.25% trypsin to completely cover the bottom of the culture flask, and incubate at 37°C with 5% CO2 for 2-3 minutes. When all cells become round and gradually detach from the bottom of the culture flask, gently tap and shake the bottom and sides of the flask, and immediately add 6 mL L MEM complete culture medium (3 times the volume of trypsin is sufficient) to stop the trypsin digestion. Repeatedly pipette the cells 5-10 times to prepare a single-cell suspension.

[0285] 6.2 Grouping

[0286] When the tumor volume reaches 50mm 3 At 18 days post-tumor cell inoculation, the melanoma model was randomly divided into six groups (n=6), with 5-6 mice in each group. The groupings are as follows:

[0287] 1) Saline group: injected with normal saline and not exposed to light;

[0288] 2) Lipo(empty)+L group: The photoresponsive drug delivery carrier (Lipo(empty)) obtained in step 1.1.4 of Example 1 was injected, which was not loaded with any drug and was exposed to light;

[0289] 3) Mixture group: A physical mixture formed by injecting the small-particle-size metal polyphenol coordination polymer micelles (DAS-NP) loaded with dasatinib obtained in step 2.1.1 of Example 2 and the photosensitive liposomes (S@Lipo) encapsulating sunitinib obtained in step 2.1.3 of Example 2, and without light exposure;

[0290] 4) Mixture+L group: A physical mixture formed by injecting the small-particle-size metal polyphenol coordination polymer micelles (DAS-NP) loaded with dasatinib obtained in step 2.1.1 of Example 2 and the photosensitive liposomes (S@Lipo) encapsulating sunitinib obtained in step 2.1.3 of Example 2, and then irradiated with light;

[0291] 5) MS@Lipo group: The photoresponsive drug delivery system (MS@Lipo) obtained in step 2.1.4 of Example 2 was injected without light exposure;

[0292] 6) MS@Lipo+L group: The photoresponsive drug delivery system (MS@Lipo) obtained in step 2.1.4 of Example 2 was injected without light exposure.

[0293] like Figure 12As shown in Figure A, the drugs were administered via tail vein injection on days 0, 2, and 4, for a total of three injections. The doses of dasatinib and sunitinib were 1.2 mg / kg and 10 mg / kg, respectively. Four hours after the tail vein injection, the groups requiring phototherapy were exposed to near-infrared light (660 nm, 300 mW / cm²). 2 Irradiate the tumor for 10 minutes.

[0294] Tumor volume and body weight were recorded every two days.

[0295] Tumor volume (mm) 3 The formula for calculating V is as follows: V = (length) × (width) 2 / 2.

[0296] Tumor growth and mouse body weight were continuously monitored in each group until the tumor volume reached 1000 mm. 3 That is, until some mice in the saline group died (day 22), at which point the mice were recorded as dead.

[0297] 6.3 Results

[0298] 6.3.1 Results of tumor volume assessment

[0299] like Figure 12 Figure B shows a tumor growth curve. The horizontal axis represents time, and the vertical axis represents tumor volume.

[0300] from Figure 12 As shown in Figure B, within 0-24 days, the tumors in the saline and Lipo (empty) + L groups exhibited rapid and continuous growth, while the Mixture and Mixture + L groups had a weak inhibitory effect on tumor growth. The MS@Lipo and MS@Lipo+L groups significantly inhibited tumor growth, with the MS@Lipo+L group showing the strongest inhibitory effect on tumor growth, reaching a tumor inhibition rate of 80%. Furthermore, the photoresponsive drug delivery system performed better than the Mixture and Mixture+L groups (i.e., a physical mixture of dasatinib-loaded metal polyphenol coordination polymer micelles DAS-NP and sunitinib-encapsulated photosensitive liposomes S@Lipo) in both untreated and treated groups. This is because the particle size difference between the polymer micelles and photosensitive liposomes in the Mixture and Mixture+L groups is too large. Polymer micelles with a particle size of 40 nm are easily cleared in the bloodstream, resulting in insufficient drug accumulation in the tumor. Moreover, the two drugs cannot achieve synchronized pharmacokinetic levels, making it difficult to exert a synergistic therapeutic effect.

[0301] 6.3.2 Results of Survival Curve Examination

[0302] like Figure 12 Figure C shows the mouse survival curve.

[0303] from Figure 12 As shown in the results, compared with the Saline group, the survival of the MS@Lipo+L group was significantly prolonged by 63.1%, and that of the MS@Lipo group was prolonged by 34.8%; the survival of the Mixture group was prolonged by 10.9%, and that of the Mixture+L group was prolonged by 19.6%; the survival of the Lipo(empty)+L group was also prolonged to some extent.

[0304] like Figure 12 Figure D shows the mouse's weight.

[0305] from Figure 12 As shown in the results, there was no significant difference in body weight among the groups of mice within 0-24 days, indicating that the photoresponsive drug delivery system (MS@Lipo) is safe for mice.

[0306] Compared to the MS@Lipo group, the MS@Lipo+L group's superior antitumor effect primarily lies in the rapid escape of sunitinib from the water cavities of the photosensitive liposomes under photoresponsive conditions and the rapid disintegration of the dasatinib-loaded polymer micelles from the photosensitive liposomes. This allows sunitinib to be released first, exerting an anti-tumor angiogenesis effect. Subsequently, the dasatinib-loaded polymer micelles penetrate deep into the tumor and enter the tumor cells via endocytosis, releasing dasatinib to antagonize the formation of VM (tumor vascular mimicry). In other words, the first drug targeting the extracellular tumor cell is released first, followed by the second drug targeting the intracellular tumor cell, thus exerting a comprehensive and effective antitumor effect through this combined internal and external approach. Existing anti-angiogenic drugs not only fail to exert their effects but also induce tumors to form more VMs more rapidly. Since VMs are insensitive to traditional radiotherapy and chemotherapy, resistance to conventional anti-angiogenic drugs caused by VMs can occur. The photoresponsive drug delivery system of this invention overcomes the resistance to anti-angiogenic drugs caused by VMs by simultaneously delivering anti-VM drugs to tumor cells, inhibiting tumor growth and thus helping to solve the problem of drug resistance. Furthermore, since anti-VM drugs and anti-angiogenic drugs target different sites, anti-angiogenic drugs are first released to extracellular targets within tumor cells, while anti-VM drugs are released to intracellular targets, allowing for effective accumulation at the tumor site and further enhancing the anti-tumor effect of the combined drug delivery. Simultaneously, the photoresponsive drug delivery system of this invention possesses photoresponsive characteristics, overcoming the limitations of relying on highly expressed enzymes or acidic microenvironments in tumor tissue for nanocarrier drug release, and is no longer affected by the heterogeneous tumor microenvironment.

[0307] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A photoresponsive drug delivery carrier, characterized in that, The invention comprises photosensitive liposomes and metal polyphenol coordination polymer micelles, wherein the metal polyphenol coordination polymer micelles include a metal polyphenol network coating and polymer micelles, and the metal polyphenol network coating is coated on the surface of the polymer micelles. The metal polyphenol coordination polymer micelles are loaded on the surface of the photosensitive liposomes; The photosensitive liposome comprises a liposome membrane and an internal aqueous phase located within the liposome membrane, the internal aqueous phase comprising ethylenediaminetetraacetic acid and an ammonium salt, and the liposome membrane comprising a lipid material and a photosensitizer.

2. The photoresponsive drug delivery carrier as described in claim 1, characterized in that, The particle size of the photosensitive liposomes is 100–150 nm. And / or, the mass ratio of the photosensitive liposomes, the metal polyphenol network coating, and the polymer micelles is (0.1–0.8):(0.05–0.4):1; And / or, the lipid material is selected from one or both of phospholipids and cholesterol; And / or, the photosensitizer is selected from porphyrin-phospholipids; And / or, the ammonium salt is selected from one or more of ammonium sulfate, ammonium phosphate, ammonium chloride, and ammonium acetate.

3. The photoresponsive drug delivery carrier as described in claim 2, characterized in that, The lipid material is a mixture of phospholipids and cholesterol, and the molar ratio of the phospholipids to cholesterol is (30-80):(0.1-60); And / or, the molar ratio of the lipid material to the photosensitizer is (60-98):2; And / or, the mass ratio of the lipid material to ethylenediaminetetraacetic acid is 1:(2-6); And / or, the mass ratio of the lipid material to the ammonium salt is 1:(2-6); And / or, the phospholipid is selected from one or more of dioleoylphosphatidylcholine, phosphoric acid choline, dioleoylphosphatidylethanolamine, and dipalmitoylphosphatidylcholine; And / or, the ammonium salt is selected from ammonium sulfate.

4. The photoresponsive drug delivery carrier as described in claim 3, characterized in that, The photosensitive liposomes were prepared using a thin-film hydration method. Preferably, the photosensitive liposomes are prepared by dissolving lipid materials and photosensitizers in a first organic solvent to form a thin film, adding ammonium salts and ethylenediaminetetraacetic acid for hydration, and obtaining the photosensitive liposomes.

5. The photoresponsive drug delivery carrier as described in claim 1, characterized in that, The polymer micelles have a particle size of 30–50 nm; And / or, the raw material of the polymer micelles comprises a polymer material selected from any one or more of polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-glycolic acid), polycaprolactone, polytrimethylene carbonate, polydioxanone, polyacrylic acid, polyacrylate and polypeptides to form a block structure polymer material. And / or, the metal polyphenol network coating is a three-dimensional network formed by polyvalent metal ions and polyphenolic compounds.

6. The photoresponsive drug delivery carrier as described in claim 5, characterized in that, The multivalent metal ions are selected from one or more of Fe(III), Mn(II), Co(II), Zn(II), Ni(II), Cu(II) and Ti(IV); And / or, the polyphenolic compounds are selected from one or more of tannic acid, ellagic acid, epigallocatechin gallate, chlorogenic acid, gallic acid, caffeic acid, quercetin, myricetin, luteolin, luteolin, resveratrol, gossypol, and proanthocyanidins; And / or, the polymer material is a block structure polymer material formed by polyethylene glycol and polycaprolactone.

7. The photoresponsive drug delivery carrier as described in claim 5, characterized in that, The polymer micelles were prepared using a nanoprecipitation method. Preferably, the polymer micelles are prepared by dissolving a polymer material in a second solvent to form an organic phase, and then adding it dropwise into an aqueous phase to obtain the polymer micelles.

8. The method for preparing the photoresponsive drug delivery carrier according to any one of claims 1-7, characterized in that, Includes the following steps: The photosensitive liposomes and metal polyphenol coordination polymer micelles self-assemble to form the photoresponsive drug delivery carrier.

9. A photoresponsive drug delivery system, characterized in that, The invention comprises a photoresponsive drug delivery carrier as described in any one of claims 1-7 and an antitumor drug, wherein the antitumor drug comprises a first drug or a second drug, and the photosensitive liposome encapsulates the first drug or the polymer micelles load the second drug.

10. The photoresponsive drug delivery system as described in claim 9, characterized in that, The first drug is selected from anti-angiogenic drugs; preferably, the first drug is selected from one or more of sunitinib, sorafenib, axitinib, and lenvatinib; And / or, the second drug is selected from drugs that inhibit angiogenesis mimicry; preferably, the second drug is selected from one or more of dasatinib, CVM-1118 and fasudil.

11. Use of the photoresponsive drug delivery carrier as described in any one of claims 1-7 or the photoresponsive drug delivery system as described in claim 9 or 10 in the preparation of antitumor drugs.

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